Bioactive implant for myocardial regeneration and ventricular chamber restoration

ABSTRACT

Bioactive implant for myocardial regeneration and ventricular chamber support including an elastomeric microporous membrane. The elastomeric microporous membrane being at least one non-degradable polymer and at least one partially degradable polymer. The non-degradable polymer is selected from polyethylacrylate and polyethylacrylate copolymerized with a hydroxyethylacrylate comonomer. The partially degradable polymer is selected from caprolactone 2-(methacryloyloxy)ethyl ester and caprolactone 2-(methacryloyloxy)ethyl ester copolymerized with ethylacrylate. The elastomeric microporous membrane further includes a nanofiber hydrogel, and cells. The bioactive implant, having one or two helical loops, contributes to the restauration of the heart conical shape. Cardiac wrapping by ventricular support bioprostheses of the present invention, having reinforcement bands spatially distributed as helicoids, recovers the sequential contraction of the myocardium resulting in the successive shortening and lengthening of the ventricles, therefore improving the ejection (systolic function) and suction of blood (diastolic function).

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of PCT/EP2011/056576, filed26 Apr. 2011, which claims the benefit of U.S. Provisional PatentApplication No. 61/327,864 filed 26 Apr. 2010 and EP 10305851.7 filed 30Jul. 2010, the entirety of which applications are hereby incorporated byreference into this application.

The present invention generally relates to the field of myocardialrepair, more particularly to a method and to a bioactive implant forrepairing myocardium and support ventricular chamber configuration andfunction.

BACKGROUND OF THE INVENTION

Heart failure (HF) is primarily a condition of the elderly, and thus thewidely recognized “aging of the population” contributes to theincreasing incidence of HF. The incidence of HF approaches 10 per 1000population after age 65 and approximately 80% of patients hospitalizedwith HF are more than 65 years old.

Heart failure is a major and growing public health problem in thedeveloped countries. In the United States approximately 7 millionpatients have HF, and more than 550 000 patients are diagnosed with HFfor the first time each year. The disorder is the primary reason for 12to 15 million office visits and 6.5 million hospital days each year.

Heart failure is the most common Medicare diagnosis-related group (i.e.,hospital discharge diagnosis), and more Medicare dollars are spent forthe diagnosis and treatment of HF than for any other diagnosis.

In Europe, the epidemiology is not well known; it is estimated thatabout 30 millions patients suffer from heart failure.

Cell transplantation and tissue engineering to the diseased heart areemerging as promising strategies to prevent or to treat refractory heartfailure that cannot successfully be treated by conventional therapies.The advances in cellular biology, in biological engineering andnanotechnologies give further advances in this option. Implantingexogenous cells supported by scaffolds in the myocardial scar tissue toreplace the damaged or the disabled cells is a safe and efficienttherapeutic approach.

Stem Cell Niche and Cell Homing

After myocardial infarction, not only the changes affect the contractileelement of the myocardium (cardiomyocytes) but also the extracellularmatrix. The collagen type 1 percentage decreases from 80% to 40%, thiscollagen is responsible with the other elements of the heart muscle ofthe normal ventricular geometry.

The efficiency of cell therapy to augment recovery after myocardialischemia depends on the sufficient recruitment of applied cells to thetarget tissue. Homing to sites of active neovascularization is a complexprocess depending on a timely and spatially orchestrated interplaybetween chemokines (e.g. SDF-1), chemokine receptors, intracellularsignalling, adhesion molecules (selectins and integrins) and proteases.

Until now, cell transplantation for cardiac support and regeneration waslimited by poor effects in ventricular function. This can be due to thelack of gap junctions between the native myocardium and the graftedcells. Also, cell transplantation seems to be limited by the relocationof transplanted cells to remote organs and noninfarcted myocardium andby the death of transplanted cells. Most cell death occurs in the firstfew days post-transplantation, likely from a combination of ischemia,apoptosis and inflammation. Apoptosis can be induced byanchorage-dependent cells detaching from the surrounding extracellularmatrix.

The cell niche, a specialized environment surrounding stem cells,provides crucial support needed for cell maintenance. Compromised nichefunction may lead to the selection of stem cells that no longer dependon self-renewal factors produced by its environment. Strategies forimproving cell survival and differentiation such as tissue engineering,has been developed.

Cardiac Tissue Engineering

Extra cellular matrix remodeling in heart failure (excessive matrixdegradation and myocardial fibrosis) contributes to Left Ventricular(LV) dilatation and progressive cardiac dysfunction. Myocardial tissueengineering should provide structural support to the heart, specificscaffolds should help to normalize cardiac wall stress in injuredregions improving strain distribution. Engineering materials requiringspecific properties of stiffness and resistance to deformation can beimplanted or seeded into the myocardial tissue. They are composed ofnatural or synthetic structure capable of supporting 3D tissueformation. Survival and engraftment of cells within the environment ofthe ischemic myocardium represents a challenge for all types of cells,regardless of their state of differentiation. Scaffolds characteristicsare critical to recapitulating the in vivo milieu and allowing cells toinfluence their own microenvironments. Such scaffolds serve at least oneof the following purposes: allow cell attachment and migration, deliverand retain cells and biochemical factors, enable diffusion of vital cellnutrients and expressed products, and exert certain mechanical andbiological influences to modify the behavior of the cell phase. Inaddition, development of gap junctions within the new created tissue aswell as with the host myocardial tissue are of great functionalinterest.

Ventricular Chamber Restoration

Restoration of ventricular shape and geometry is a surgical proceduredesigned to restore or remodel the left and/or right ventricle to itsnormal, conical shape and size in patients with akinetic segments of theheart, secondary to either post infarction cardiomyopathy or dilatedcardiomyopathy. The restoration procedure can be performed during orafter coronary artery bypass grafting (CABG), mitral valve repair orreplacement and other procedures such as implantation of stem cells formyocardial regeneration. Surgical ventricular restoration has beenperformed: 1) by partial resection of the ventricular wall using cardiacarrest and cardiopulmonary bypass (extracorporeal circulation), or 2) byexternal ventricular remodelling, e.g. cardiac wrapping with autologoustissues like the latissimus dorsi muscle flap. Ventricular restorationprocedure with bioactive implants avoids cardiac arrest andextracorporeal circulation.

Ventricular Restraint Therapies

Heart failure patients develop oversized, dilated hearts due toincreased filling pressures. Over time the increased workload of theheart can lead to a change called remodeling, which is the enlargementand thinning of the ventricles. The failing cardiac muscle need to besupported to decrease the ventricular wall stress. Mesh wrap devicesthat are implanted around the heart have been used. These devices areintended to prevent and reverse the progression of heart failure byimproving the heart's structure and function, leading to improvements inthe survival and quality of patient's life. For example, implantabledevices have been tested for ventricular restraint therapy, likepolyester netlike sack designed for placement around the heartfabricated into a multifilament mesh knit (C or Cap device, Acorn). Alsoa nitinol mesh for ventricular wrapping was investigated (HeartNetdevice, Paracor). Permanent implantation experience of both devicesshowed adverse effects like restriction in diastolic function and lackof improvement of systolic function, without evidence of myocardialhealing. These results have limited its large clinical application,including the “not to approve” U.S. Food and Drug Administration (FDA)decision.

Translational Research

Experimental and clinical studies have been performed on stem celltherapy and tissue engineered approaches for myocardial support andregeneration. The results of these investigations tend to demonstratethe interest of simultaneous intrainfarct stem cell therapy with thefixation of cell-seeded matrices onto the epicardium of infarctedventricles.

Experimental studies suggest that simultaneous autologousintramyocardial injection of stem cells and fixation of a cell-seededcollagen matrix onto the epicardium is feasible. However, the long-termefficacy of this approach is compromised by the complete biodegradationof the grafted collagen matrix.

WO2006/036826 discloses a tissue-engineering scaffold containingself-assembled-peptide hydrogels.

US2005/0095268 describes a cardiac wall tension relief with cell lossmanagement.

The article of Boublik et al. (Tissue engineering, 2005) relates to themechanical properties and remodelling of hybrid cardiac constructs madefrom heart cells, fibrin, and biodegradable, elastomeric knitted fabric.

In summary, the following problems are encountered in the field ofmyocardial repair.

1) It is difficult to repair a large myocardial scar.

2) Cell bio-retention and engraftment within scar tissue is too low.

3) Mortality of implanted cells in ischemic myocardium is high.

4) Extracellular matrix remodeling in ischemic heart disease (excessivematrix degradation and myocardial fibrosis) contributes to LV dilatationand progressive cardiac dysfunction.

5) The therapeutic limitation of heart dilatation and the recovery ofthe native elliptical shape of ventricular chambers are key prognosticfactors for survival in HF patients.

6) In cell transplantation, survival and engraftment within theenvironment of the ischemic myocardium represents a challenge for alltypes of cells, regardless of their state of differentiation.

7) Up to now, the optimal cell-matrix combination for robust andsustained myocardial restoration has not been identified.

8) The long-term efficacy of the approach—autologous intramyocardialinjection of stem cells and fixation of a cell-seeded collagen matrixonto the epicardium—is compromised by the complete biodegradation of thegrafted collagen matrix.

9) There are undesired effects of growth factor administration.

10) Tissue viability/evolution over time.

SUMMARY OF THE INVENTION

The present invention provides a bioactive implant for repairingmyocardium and support ventricular chamber configuration and function,and a method for preparing such implant. The bioactive implant isgrafted onto and/or into the ventricular wall for myocardialregeneration, for left or right ventricular support and to restore theelliptical shape of ventricular chambers.

The scaffolds are created by the combination of a membrane which is amix of biodegradable materials (biological or synthetic) withnon-biodegradable (biostable) synthetic materials, with hydrogel andcells. During the procedure, cells, e.g. stem cells mixed with hydrogel,are seeded into/onto the membrane, i.e. a template form, and immediatelyor secondarily grafted onto diseased myocardial tissue.

The method of the invention comprises the steps of creating a scaffoldcombining biodegradable with non-biodegradable materials, obtainingautologous cells or cells from a donor, implanting the cells into thematrix and grafting the composite cellular scaffolds onto the heart.

The implant and method of the present invention aims to improveventricular function, to limit chronic dilation of ventricular chambersand to restore the native elliptical shape of the heart as a newmodality in the treatment of heart failure.

The advantages of the objects of the present invention are thefollowing:

a) Stem cell transplantation induces myocardial angiogenic and/ormyogenesis improving myocardial viability and reducing scar fibrosis.

b) Matrix scaffolds grafting improves stem cell niche and cell homing,consequently increasing the thickness of the infarct scar with viabletissues. This composite material helps to normalize cardiac wall stressin injured regions. In addition, new vessels formation from theepicardium and from the surrounding well irrigated myocardium contributeto the reduction of the fibrosis and size of infarction scars, inducingthe regeneration of contracting cells and extracellular collagen matrix.

c) Synthetic cardiac support material onto the heart brings long-termbeneficial impact on ventricular chamber size and shape reducing tensionand promoting limitation of adverse remodelling. In addition, thismaterial helps to normalize cardiac wall stress in injured regionsimproving strain distribution, avoiding scar dyskinesia and the risk offormation of ventricular aneurysms, ventricular wall rupture and mitralvalve insufficiency.

d) Adapted ventricular wrapping. The bioactive implants of the presentinvention are designed for left ventricular and/or right ventricularsupport and regeneration, including different sizes for partial orcomplete ventricular wrappings. The implant characteristics (mechanical,physical, chemical, biological) are adapted for the left or the rightventricle geometry, physiology and pathology.

e) Maintenance and survival of the implanted cells in situ. Preparationand maintenance of the cellular population of the bioactive implants isobtained by cardiac cell therapy before, during or after grafting“Bioactive Implants” onto the heart. Cell transplantation is performedusing either catheter-based approaches via the endocardium(endoventricular), via an intravascular procedure (through coronaryarteries or veins) or injecting the cells through the epicardium duringcardio-thoracic surgery, thoracoscopy or computer-robotic assistedprocedures. Additionally, lowered oxygen tension (e.g., 5% to 15%) isused during cell growth as a preconditioning procedure to improve cellsurvival following patch implantation in is chemic myocardium.

f) Prevention of LV dilatation and progressive cardiac dysfunction.According to an aspect of the invention, the entire organ is containedwith the elastomeric membrane of the bioactive implant to prevent heartdilatation. Thus, with decreased ventricular wall tension thecomplementary treatment of grafting biological tissue (e.g. peptides andstem cells) can successfully achieve myocardial regeneration.Additionally, pacing electrodes can be used in the method of theinvention and also incorporated into the Bioactive Implants and thenative myocardium for synchronous electrostimulation of the implantedtissue and other electrophysiological treatments (defibrillation,resynchronization, etc).

g) Regenerative treatment in association with implants in view ofsurvival and engraftment. Cell-based myocardial regenerative treatmentscan be associated, i.e. intramyocardial and intrainfarct stem celltransplantation, with the implantation of bioactive implants onto theheart. Associated method for seeding or implanting stem cells into oronto the Bioactive Implants using the following methods: mechanical(shaking, centrifugation), chemical (electrophoresis), physical(electroporation), etc. Seeded or implanted cells that can beangiogenic, cardio-myogenic or pluripotents. Additionally, BioactiveImplants can be labelled with products (dies, microspheres,radioisotopes, iron-particles, etc) for evaluation of biodegradation,integration, proliferation, differentiation and function, usingradiologic, ultrasound-echocardiographic, radioisotopic, metabolic(PET), RMI, CT Scan and bio-luminescence-fluorescence methods (etc.).

h) Adjusted composition of the bioactive implants. The composition ofthe bioactive implants has a percentage of non-biodegradable (synthetic)versus biodegradable (biological or synthetic) components, which rangesfrom 10% to 90%.

i) To obviate the undesired systemic effect of growth factors, thesynthetic material is designed to locally release angiogenic factorssuch as VEGF, HBEGF, bFGF. Additionally, according to an embodiment ofthe present invention, Bioactive Implants are endowed with a system forthe controlled release of angiogenic and antiapoptotic factors.

j) Assessment of the tissue growth and viability. Sensing Electrodes areincorporated in the bioactive implants and connected to a bioelectricalimpedance measuring device. The goal of this implantable monitor is toassess by telemetry the evolution of engineered tissue in cardiacregeneration and to detect early pulmonary oedema in heart failurepatients.

The invention will be more fully described by reference to the followingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic diagram of a bioprosthesis for biventricularheart failure patients.

FIG. 1B is a schematic diagram of a bioprosthesis for right ventricularheart failure patients.

FIG. 1C is a schematic diagram of a bioprosthesis for left ventricularheart failure patients.

FIGS. 2A and 2B are schematic diagrams of a single helical loop used toreinforce the bioprosthesis.

FIG. 3 is a schematic diagram a double loop used to reinforce thebioprosthesis.

FIGS. 4A and 4B are schematic diagrams of a bioactive patch.

FIG. 5 of the bioprosthesis fixed with epicardial interrupted sutures.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

The present invention provides a bioactive implant for myocardialregeneration and cardiac support.

The bioactive implant of the invention constitutes a scaffold.

As used herein, a scaffold is a material acting as a template for cellsto grow and produce new tissue. The scaffold of the invention can beprovided in a desired size and shape dependent on the contemplated use.Indeed each ventricle has different wall thickness, wall tension andchamber pressure. To reply to the variety of size and shape, the implantof the invention can notably be provided in the form of a cone or in theform of a square depending on the intended and location use. It can beof a size of from 1 cm² to 20 cm². As the bioactive construct of theinvention is implanted in the body of a mammal, for example a human, thematerial of the membrane is chosen to be physiologically inert to avoidrejection or other negative inflammatory response.

According to the present invention, the bioactive implant comprises

-   -   I. an elastomeric microporous membrane (patch) comprising at        least:        -   a. one non-degradable synthetic polymer and        -   b. one partially degradable synthetic polymer    -   II. a peptide nanofiber hydrogel and    -   III. cells.

As used herein, a membrane is a material having one of its threedimensions (its thickness) much smaller than its other two dimensions(its length and width), these latter being comparable in magnitude. Theterm patch may be used equivalently instead of the term membrane. Anelastomer is a crosslinked macromolecular material which in workingconditions is above its glass transition temperature and thus is able torapidly recover its original unstressed dimensions after cessation ofmechanical loading not exceeding a critical value. An elastomericmembrane is a membrane made out of an elastomer. A microporouselastomeric membrane is an elastomeric membrane in which the elastomerconfigures a system of interconnected void spaces, the pores, throughoutthe bulk of the membrane, the pores having linear dimensions in therange of several tens to a few hundreds of micrometers. The pores areintended to host cells and the extracellular matrix produced by them.The pores of the microporous elastomeric membrane may also be filledwith the peptide nanofiber hydrogel. Alternatively or additionally, thepeptide nanofiber hydrogel may be placed on top of the elastomericmembrane. The microporous membrane of the invention configures athree-dimensional environment able to convey mechanical stimuli to thecells and to facilitate three-dimensional cell-to-cell interactions.

According to the present invention, the porosity of the membrane iscomprised between 70% and 90%, said pores being interconnected andhaving preferred diameters comprised between 50 microns and 500 microns,for example between 80 microns and 150 microns.

As used herein, a polymer is a macromolecule consisting in the repeat ofa few different units (if one, a homopolymer; if more than one, acopolymer). A synthetic polymer is a polymer not present naturally inbiological system. A non-degradable polymer is a polymer which remainschemically unaltered in vivo. The term biostable and the termnon-degradable may be used interchangeably. A degradable polymer is apolymer which in vivo undergoes depolymerization (scission) reactionswhose products can be toxic or non-toxic, metabolized or non-metabolizedby the tissues or organs of the host. The term biodegradable, the termdegradable and the term bioresorbable may be used interchangeably. Theterm biodegradable refers to material that degrade or break down, withtime, upon interaction with a physiological environment into componentsthat can be metabolised or excreted by the body. A partially degradablepolymer is a polymer composed of at least one non-degradable polymer andat least one degradable polymer.

More specifically, the non-degradable or biostable synthetic polymer a.is selected from the group consisting of polyethylacrylate orpolyethylacrylate copolymerized with a 10% wt or a 20% wthydroxyethylacrylate co-monomer. Scaffolds made of this polymer areadvantageously produced by the template leaching method, using astemplate an arrangement of sintered spheres and/or fiber fabrics. Thepartially degradable synthetic polymer b. is selected from the groupconsisting of caprolactone 2-(methacryloyloxy)ethyl ester orcaprolactone 2-(methacryloyloxy)ethyl ester copolymerized withethylacrylate in weight proportions of this last co-monomer comprisedbetween 30% and 80%. Using caprolacton as a component of the partiallydegradable polymer is particularly advantageous. Indeed sincecaprolacton is produced by chemical synthesis, it does not produceantigenic reaction, by comparison with collagen.

Also the degradation of a part of the implant reduces a possible risk ofchronic restriction of the diastolic function. Indeed, all prostheticmaterials implanted around the heart usually produce chronicinflammatory reaction resulting in fibrosis, responsible for therestriction of the diastolic function. The amount of fibrosis is relatedwith the characteristics and the amount of the implanted material. Usinga bioactive implant according to the present invention, that is to sayan implant containing a biodegradable portion, allows a decrease of theinflammatory reaction with time and thus reduces the possible risk of arestriction of the diastolic function.

Also, it is to be noted that the scaffold membrane of the invention ismade of a combination of degradable polymers and partiallynon-degradable polymers.

The bioactive implant provides a suitable environment for cell homing,growth and differenciation (myocardial repair), as well as mechanicalsupport to the heart. The combination of degradable polymers andpartially non-degradable polymers is advantageous because cellsimplanted in niches will organize, connect and contract more easily withtime if they are surrounded by material that degrade with time and ifnot directly surrounded by a synthetic prosthetic material. However somedefinitive prosthetic fibres are necessary to avoid progressive heartdilatation. The combination of both polymers, degradable andnon-degradable ones, allows a good cells implantation and to keep thescaffold structure.

This polymer is advantageously produced by the template leaching method,using as template an arrangement of sintered spheres and/or fiberfabrics. The percentage of non-degradable polymers versus degradablepolymers is comprised between 10 and 90% wt; it is preferably comprisedbetween 10 and 48% wt. The membrane of the present invention is thus acombination of a degradable component and a non-degradable component.Although the membrane comprises at least one polymer that is partiallybiodegradable, the implant made from such membrane (and thus from suchpolymer) must maintain the structural integrity for a time required forthe intended use.

The membrane of the invention may additionally comprise biomaterials ofnanoporous or nanoscale fiber dimensions, for example a coating ofhyaluronic acid, independently from the composition and presence of thehydrogel.

As used herein, a hydrogel is a macromolecular material, by which eitherphysical or chemical crosslink interactions produce a macromolecule basecomponent, able to retain large amounts of water molecules. A nanofiberhydrogel is a hydrogel made of nanoporous or nanoscale fibers thatpercolates above a defined concentration to form a network.

According to an aspect of the invention, the nanofiber hydrogel can bedegradable, biologically or chemically, or non-degradable. In certainembodiments, the hydrogel includes natural molecules such as protein,peptide, oligosaccharide, polysaccharide, or proteoglycan derivedmatrices such as collagens, fibrins, alginates, chitosans, hyaluronicacid, and/or any synthetic molecule that will develop into a nanofibernetwork with gel/hydrogel properties, such a peptide nanofiber hydrogelscaffold; a class of self-complementary amphiphilic peptides thatself-assemble into nanofibers illustrates such peptides. The followingpeptide AcN-RADARADARADARADA-COONH₂ commercially available by the nameof PURAMATRIX is an example of this peptide class.

In an aspect of the invention, the nanofiber hydrogel comprises at leastone self-assembling peptide (SAPs).

A self-assembling peptide is a peptide with self-complementaryproperties able to undergo spontaneously a phase transition from adisorder sol state to a more ordered state, where the final orderedstate consists of a crystal-like structures or a collapsed amorphousmaterial. The transition is triggered by environmental parameters suchas a pH or pK threshold, temperature, etc. A self-assembling peptide gelis the spontaneous assembly of self-complementary peptides developinginto ordered chain or domains with elongated shapes and dimensions inthe range of a few to tens of nanometers, and are thus referred to asnanofibers. An example of SAP is RAD16-I. Other examples are: RAD16-II(AcN-RARADADARARADADA-COONH₂) and KLD12 (AcN-KLDLKLDLKLDL-COONH₂.

In a preferred embodiment, the nanoporous or nanoscale fiber hydrogeleither completely fills the pores of the elastomer membrane or partiallyfills the pores by forming a layer coating to the inner surfaces of themembrane's pores.

In a specific aspect of the invention, the membrane is surface-treatedto graft adhesion molecules such as functional peptides like as RGDpeptides (Arg-Gly-Asp), functional sugars or lipids, and proteins suchas laminin or laminin fragments.

The bioactive implant of the invention is designed to feature mechanicalproperties to be elastic enough to match the myocardiumcontraction-distraction activity to allow deep structural and functionalbio integration.

The implant of the invention has an elastomeric membrane which containsthe entire organ to prevent heart dilatation (i.e. a decreasedventricular wall tension).

The bioactive implant, in a particular aspect of the invention,additionally comprises a system for the controlled release or absorbanceof active molecules such as any organic molecule, such as smallmolecule, peptide, lipid, sugar, protein, proteoglycan, with angiogenic,antiangiogenic, pro-regenerative, anti-regenerative, apoptotic,necrotic, antiapoptotic and antinecrotic activity, such as VEGF, IL-6,IL-10, IGF-1, FGF-2, HBEGF, bFGF and chitosan.

Chitosan, a natural polymer of glucosamine and N-acetyl glucodamine, iswidely used in the pharmaceutical and tissue engineering fields due toits biocompatibility, biodegradability, and antimicrobial properties.Addition of chitosan improves the physical properties of bioactiveimplants, and enhances their ability to support endothelial cells andangiogenesis for use in cardiovascular tissue engineering applications.

The release or absorbance system may consist in:

(a) the molecule encapsulated in degradable microparticles made of suchpolymers as chitosan, hyaluronic acid, complexes of these last twopolymers, or a degradable polyester, such as polyglycolic acid, orpolylactic acid, or polycaprolactone; the said microparticles embeddedin the gel filling or coating the membrane's pores;

(b) the molecule included in the gel filling or coating the membrane'spores associated non-specifically or specifically to the structure ofthe gel filling material;

(c) the molecule covalently or non-covalently bonded to theself-assembling peptide filling or coating the membrane's pores.

(d) the molecule with absorbance capacity to eliminate any organicmolecule with antiangiogenic, anti-regenerative, apoptotic or necroticactivity. The bioactive implant of the invention, in another aspect,additionally comprises cytokines and angiogenic antiapoptotic peptides.

It has binding capacity of components secreted by the necrotic tissue.Accordingly, it has the ability to modulate and neutralize the effect ofcomponents such as Midkine (MDK), a negative regulator of angiogenesis.

The implant of the invention can be functionalized with biologicalactive motifs (peptides and glycopeptides) to promote cellularresponses, in particular, myocardial instruction to maintain phenotype,allow cell-cell contact and establishment of gap-junctions.

The bioactive implant of the invention can be designed for elliptical orconical heart shape restoration, in which both ventricles are completelywrapped by the device. The structure of this device consists in specialreinforcements at the level of the anatomical bands, forming two helicalloops of “non-degradable polymers” for conical shape restoration. Thegeometric disease in ischemic dilated cardiomyopathy is the sphericalchamber, which is different than the elliptical or conical normal heartshape. The use of two helical reinforcement bands allows athree-dimensional recovery of the original ventricular elliptical shape.

The bioactive implant of the present invention comprises cells.

The cells can be myogenic or cardiomyogenic cells. According to thisembodiment, the cells are selected from the group consisting of skeletalmyoblasts, smooth muscle cells, fetal and neonatal cardiomyocytes, adultventricular cardiomyocytes, cardiospheres and epicardial progenitors.

The cells can alternatively be angiogenic cells, such as bone marrow andperipheral blood mononuclear fraction, bone marrow and peripheral bloodendothelial progenitors, endothelial cells, mesothelial cells fromomentum, adipocyte derived stem cells, stem cells from adiposeepicardial tissue and multipotent menstrual blood stromal cells.

The cells can also be pluripotent stem cells. In this embodiment, thecells are selected from the group consisting of embryonic cells, animalembryonic cells, adult stem cells, fetal stem cells, neonatal stemcells, non-human stem cells, umbilical cord cells, induced pluripotentstem cells (iPSCs), bone-marrow mesenchymal stem cells (MSCs), adulttestis pluripotent stem cells and human amniotic fluid stem cells(hAFSCs). In this regard the bioactive implant is designed to hosteducated or trained cells expected to grow, multiply, differentiate andorganize into a nanoscale fiber within the microporous structure of thepatch and to connect with the native myocardium. The combination of theelement of the bioactive implant along with the cells is such that itpresents a decreased ventricular wall tension and can successfullyachieve myocardial regeneration.

According to an aspect of the invention, a mixture of cells is used ascomponent of the bioactive implant. These cells can for example beselected among the following: myogenic cells, cardiomyogenic cells,angiogenic cells and pluripotent stem cells, with the above-givendefinitions.

Suitable sources of cells for bioactive implant seeding and intrainfarctinjection will depend on the types of diseases to be treated. For recentmyocardial infarction, angiogenic cells that reduce myocardial necrosisand augments vascular blood flow will be desirable. For chronic heartfailure, cells that replace or promote myogenesis, reverse apoptoticmechanisms and reactivate dormant cell processes will be useful. Forchronic ischemic cardiomyopathy, both angiogenic and cardiomyogeniccells will be associated.

According to the present invention, it is possible to embed cells in a3-dimensional structure replicating the extracellular matrix, which iscrucial for full tissue restoration and prevention of ventricularremodeling. The clinical translation of cell therapy requires avoidanceof potentially harmful drugs and cytokines, and rapid off-the-shelfavailability of cells. The combination of pre-differentiated cellswithin a functionalized scaffold, locally releasing molecules tailoredto promote in situ completion of differentiation and improve homing,survival, and functions, circumvents the potential undesired systemiceffects of growth factor administration and improve tissue restoration.

The cells seeded into matrix scaffold and supported by a syntheticventricular support device treated with adhesion molecules amelioratefunctional recovery of infarcted hearts and improve long-term evolutionby providing myocardial regeneration and gentle support.

The cell-matrix combination associated with a ventricular constraintnon-absorbable material such as, mesh cardiac wrap, positioned over thediseased myocardium improves ventricular function and reduces adversechamber remodelling.

The present invention combines a regenerative biological approach with aprosthetic cardiac support device. Stem cells associated with a tissueengineered matrix scaffold and combined with a mesh cardiac wrap shouldreduce post-ischemic fibrosis and assist the recovery of myocardialviability and compliance. This procedure can be proposed for thetreatment of ischemic heart disease, associating a regenerativebiological approach with a prosthetic support device.

The present invention constitutes a unique platform for engineeringhighly efficient contractile tissues and enhancing cell therapy.

For the present invention, the cells can be obtained from any suitablesource. They can be purchased or they can be isolated from a suitablesource by methods well known to those skilled in the art. They may becultured according to methods known to those skilled in the art. Forexample, the cells can be added to culture medium which may additionallycomprise growth factors, serum, antibiotics or any of a variety of cellculture components known to those skilled in the art.

Ischemic cardiomyopathy induces geometric alteration of the ventricularcavity, which changes from an elliptical to a spherical shape. Thegeometric disease in ischemic dilated cardiomyopathy is the sphericalchamber, which is different than the elliptical or conical normal heartshape. The sphericity index quantifies this geometric form alteration bycomparing the transverse ventricular (short) and long axis; an ellipsehas a 0.5 ratio (the length is twice the width) and a sphere is 1.0 dueto similar transverse and longitudinal dimensions.

According to one embodiment of the invention, the structure of thebioactive implant of the present invention consists in specialreinforcements at the level of the anatomical bands, forming two helicalloops of non-degradable biostable synthetic polymers. This embodiment ofthe present invention is intended to cover both ventricles for ischemicdilated cardiomyopathy. Helical ventricular artificial bands for LVelliptical shape restoration using bioactive implants is useful in thecontext of the biosurgical strategies indicated to manage patients withadvanced myocardial diseases. An important advantage of this techniqueis the fact that the surgery is performed without the risk of openingthe ventricular chambers, i.e. without extracorporeal circulation.

In another aspect, the present invention deals with a method forpreparing the bioactive implant of the invention, comprising the stepsof filling an elastomeric microporous membrane with a nanofiberhydrogel, so as to obtain a bioactive construct. In this respect, thefilling is for example made by placing a syringe into the membrane, saidsyringe being filled with the gel and gently evacuating the air in thepores; according to another step, said construct is cultured underbiophysical, mechanical conditions (i.e. compression and elongation);then a step of seeding or implanting cells onto or into said bioactiveconstruct using the following methods: mechanical (shaking,centrifugation), chemical (electrophoresis), physical (electroporation).The cells that can be used are myogenic, cardiomyogenic, angiogenic orpluripotent stem cells.

Another method for preparing the bioactive implant of the inventioncomprises the steps of obtaining cells, for example, myogenic,cardiomyogenic, angiogenic or pluripotent stem; culturing said cells invitro; mixing said cells with a nanofiber hydrogel; and filling anelastomeric microporous membrane with said cell-containing nanofiberhydrogel, so as to obtain a bioactive construct. The method of theinvention may also comprise the step of culture of said bioactiveconstruct under local in vitro electrostimulation.

In a specific aspect of the method, the cells are cultured under loweredoxygen tension.

In a still specific aspect of said method, the bioactive implant iscultured so as to be adapted to left ventricular and/or rightventricular support and regeneration, for partial or completeventricular wrappings.

The present invention also deals with a method for repairing themyocardium of an individual comprising the steps of preparing abioactive implant according to the invention and implanting thebioactive implant into and/or onto the myocardium.

In a more specific aspect, the method for repairing the myocardiumcomprises an additional step consisting in injecting cells through theepicardium during cardio-thoracic surgery, thoracoscopy orcomputer-robotic assisted procedures. The injected cells can beautologous stem cells cultured in hypoxic conditions.

The present invention also deals with an elastomeric microporousmembrane, comprising at least one non-degradable polymer, at least onepartially degradable polymer, and at least one biomaterial of nanoporousor nanoscale fiber dimensions, said membrane having a porosity comprisedbetween 70% and 90%, the pores being interconnected and having diameterscomprised between 50 microns and 500 microns, for example between 80microns and 300 microns, preferably between 80 microns and 150 microns,wherein

a. the non-degradable polymer is selected from the group consisting ofpoly(ethylenglycol diacrylate), polyethylacrylate and polyethylacrylatecopolymerized with a 10% wt or a 20% wt hydroxyethylacrylate comonomer;and

b. the partially degradable polymer is selected from the groupconsisting of polycaprolactone, caprolactone 2-(methacryloyloxy)ethylester and caprolactone 2-(methacryloyloxy)ethyl ester copolymerized withethylacrylate in weight proportions of this last comonomer comprisedbetween 30% and 80%,

wherein the percentage of non-degradable polymers versus degradablepolymers is comprised between 10% wt and 90% wt.

This membrane may be used in a bioactive implant for myocardialregeneration and ventricular chamber support.

In a more specific aspect, the elastomeric microporous membrane can besurface-treated to graft adhesion molecules. The adhesion moleculesbeing selected from the group consisting of functional peptides such asRGD peptides, functional sugars or lipids, and proteins such as lamininor laminin fragments.

The present invention also deals with a method for surgical myocardialrepair, comprising the steps of:

a) mixing cells with a nanofiber hydrogel,

b) positioning the elastomeric microporous membrane of the invention atthe intended location of the body, and

c) injecting or spreading the mix obtained in step a) into or onto thepositioned elastomeric microporous membrane.

The present invention also concerns a method for surgical myocardialrepair, comprising the steps of:

a) mixing cells with a nanofiber hydrogel,

b) injecting or spreading the mix obtained in step a) into or onto theelastomeric microporous membrane of the invention, so as to obtain abioactive implant, and

c) positioning the bioactive implant of step b) at the intended locationof the body.

FIG. 1A-1C illustrate adapted ventricular wrapping. FIGS. 1A-1C showdevices for complete ventricular wrapping. FIG. 1A is used forbiventricular heart failure patients. Bioprosthesis 10 is made of thesame material for both right ventricle 12 and left ventricles 14.Bioprosthesis 10 can be formed of the elastomeric microporous membrane.FIG. 1B is used for right ventricular failure patients. Bioprosthesis 20is made of high rate [60 to 80%] of biodegradable material in left heartside 14 and the elastomeric microporous membrane on the right heart side12. FIG. 1C is used for left ventricular failure patients. Bioprosthesis30 is made of high rate of biodegradable material in right heart side 12and the elastomeric microporous membrane on the left heart side 14.

Bioprosthesis 10, 20, 30 can be reinforced by helical loops made ofnon-degradable or semidegradable polymers. These materials can be thefollowing: polyethylacrylate (PEA) copolymerized withhydroxyethylacrylate comonomer, semi degradable methacrylate-endcappedcaprolactone (CLMA), polyethylene terephthalate (PET), polypropylene,polydioxanone, polyglecaprone, e-caprolactone, poly-L-lactide polymers,poly salicylic acid polymer, poly desaminotyrosyl-trypsine ethyl esterpolymer, polycarbonate urethane, polyurethanes, poly(glycerol sebacate)(PGS), elastin, silk.

Loops are made of a band of 30 mm to 40 mm width with 1 mm to 2 mmuniform thickness, or made with thickness progressively increased from 1mm to 3 mm

Helical loops follow the anatomical heart configuration, where muscularventricular bands begin at the insertion of the pulmonary artery in theright ventricle and ending at the aortic valve annulus (LV). The role ofmyocardial band is to limit ventricular dilatation, preservingelliptical shape, and contribute to systolic contraction and diastolicfilling (suction mechanism).

In one approach, a helical loop can be integrated into the ventricularbioprosthesis structure during manufacturing.

In another approach, a helical loop is a complement of a bioactive patchfixed onto a myocardial pathologic zone, during surgery or thorascocopicapproach.

In another approach, a helical loop is fixed around the heart as acomplement of the ventricular support bioprosthesis, implanted to coverthe ventricles during surgery or thorascocopic approach.

In another approach, a helical loop can be used as a single therapeuticprocedure.

For moderate heart dilatation, single apical loop 50 is used to wrapventricles, starting at the level of the left atrial appendage 52 andending at the aortic root 53, as shown in FIG. 2. (See FIG. 2B showingthe position of the left atrial appendage 52).

For severe heart dilatation, double basal and apical ventricular helicalloops 60 a, 60 b are used, starting at the level of pulmonary arteryroot 54 and ending at the level of the aortic root 53, as shown in FIG.3. Fixation of the loop/band onto the heart, onto the bioactive patchand onto the ventricular support bioprosthesis can be made by surgicalsutures and/or surgical clips and/or glue of biological or syntheticorigin.

The method for implantation of small bioactive patches or large cardiacsupport bioprostheses can be performed during cardio-thoracic surgery,thoracoscopy or computer-robotic assisted procedures. After gainingaccess, the pericardium is open to expose the heart.

In a bioactive patch implantation procedure, scaffold 70 is positionedonto the pathologic myocardial lesion, for example covering the infarctand peri-infarct zones. It is fixed to the epicardium by singleinterrupted sutures 72 (4-0 or 5-0) and covered by the pericardium, asshown in FIGS. 4A and 4B.

In ventricular support bioprothesis implantation procedure, for a devicechoice, the size of the heart is measured by a circumferential tape.Adequate size of bioprothesis 10, 20, 30 is chosen and placed aroundright ventricle 12 and left ventricle 14, as shown in FIG. 5.Bioprosthesis is placed around the ventricles by sliding it gently intoposition (arrow), from the apex of the heart to the atrio-ventriculargroove Bioprothesis 10, 20, 30 is fixed with epicardial interruptedsutures 82 (4-0) to heart 80 at the level of the A-V groove, starting atthe most posterior location. For example, fixation sutures are placedevery 2 cm to 3 cm.

The sequential contraction of the ventricular myocardium results in thesuccessive shortening and lengthening of the ventricles. These movementsmay determine the ejection and suction of blood, respectively. The shapeand duration of ventricular filling/emptying mechanism can be comparedto a stroke action induced by a piston water pump. Surgicalinterventions for heart failure like reduction ventriculectomy have notproven surgically efficacious. Removal of apical or basal ventricularsegments and the muscle bands seems to interfere with the naturalsequence of myocardial contraction and diastolic filling. Cardiacwrapping by ventricular support bioprostheses of the present invention,having bands spatially distributed as helicoids, is an advantageousphysiological therapeutic method.

EXAMPLES Example 1 Biological Evaluation of Elastomeric ScaffoldMembranes

Quantification of Cell Proliferation.

MTT Assay

The MTT system is a simple, accurate, reproducible means of measuringthe activity of living cells via mitochondrial dehydrogenase activity.The key component is 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide or MTT. Solutions of MTT solubilised in tissueculture media or balanced salt solutions, without phenol red, areyellowish in color. Mitochondrial dehydrogenases of viable cells cleavethe tetrazolium ring, yielding purple MTT formazan crystals which areinsoluble in aqueous solutions. The crystals can be dissolved inacidified isopropanol. The resulting purple solution isspectrophotometrically measured. An increase in cell number results inan increase in the amount of MTT formazan formed and an increase inabsorbance.

Material and Method

Pieces of elastomeric microporous membranes of polyethyl acrylate(PEA100 in what follows) and a copolymer of ethyl acrylate andhydroxyethyl acrylate with a 90:10 mass ratio of both monomers(hereafter PEA90) were employed. The membranes had been cut into piecesof dimensions 25 mm×25 mm, with an approximate thickness of 1.0 mm(PEA100 and PEA90A) and of 0.7 mm (PEA90B). The porosity of the membranewas 70%. The pores of the membranes consisted in layers of orthogonalfamilies of parallel cylindrical pores, with pore diameter of 150microns and pore separation of 300 microns. They were generated byletting the precursors of the polymers react inside a mould with atemplate of layers of porogen orthogonal fibers, and dissolving thetemplate afterwards to give place to said pores.

Scaffolds Conditioning Protocol

Due both to their hydrophobic nature and to their microporous structure,the elastomeric membrane needs to be pre-hydrated before cell seeding.The conditioning procedure consists in a 24 h immersion in a PBSsolution. Vacuum might be necessary to improve fluid penetration intothe pores, putting the sample in a tube sealed hermetically with a cappierced by a syringe's needle, and performing vacuum with the syringe.The pre-hydrated sample is then immersed in the culture medium. If thepH changes, the medium is renewed until the reference pH value remainsstable.

Cell Seeding

Bone marrow mesenchymal stem cells (BMC) were isolated under sterileconditions from femur and tibia bones of Wistar rats. After 2 weeks ofin vitro cultures in DMEM complete medium with L glutamine, sodiumpyruvate and 15% Fetal Bovine Serum (1^(st) passage), 10,000 cellsdiluted in 0.5 ml medium were seeded into PEA90 and PEA100 scaffolds andinto 3D Collagen type I matrix (n=5 for each sample). After careful cellseeding using a micropipette, elastomeric scaffolds and collagen matrixwere maintained 20 minutes without motion, to start cell adhesion. Atthe following step, and in order to promote a regular distribution ofBMC into the matrix pores, Petri dishes containing the elastomeric andcollagen scaffolds/matrices were shaken continuously for 10 minutes at80 g using an Orbital Shaker (Stuart Scientific, Stone, Staffordshire,UK). Afterwards cell seeded scaffolds were incubated one hour at 37° C.Finally DMEM complete medium was added to the Petri dish and the cellseeded scaffolds/matrix were cultured during 3 weeks at 37° C., 5% CO₂.

Quantification of Cell Propagation

Cultures were removed from the incubator into a laminar flow hood. Thesupernatant was removed and then the scaffolds were washed with PBS twotimes. The scaffolds were transferred into new tubes (15 ml Falcon).Aseptically the MTT solution was added in an amount equal to 10% of theculture volume (1800 microliter phenol red free medium+180 microliterMTT) and cultures were incubated for 3 hours at 37° C. in a 5% CO2humidified atmosphere. Two ml of solubilisation solution or solvent wereadded and then vortexed for 5 min. This provoked the release from thescaffold of MTT which was actively reduced by viable cells acquiring ayellow colouring. Each sample was centrifuged at 15,000 g for 5 min andthe supernatant was read at 570 nm using a multiwell spectrophotometer.

Results

Spectrophotometer assessments showed optical density (OD) values of0.13+/−0.02 for collagen matrix; 0.22+/−0.04 for PEA90A scaffolds;0.11+/−0.03 for PEA90B scaffolds; and 0.34+/−0.05 for PEA100 scaffolds.

These results showed that cell proliferation was well developed in theelastomeric scaffolds, presenting a better proliferation than the 3Dcollagen scaffolds. Until now collagen scaffolds have been used inexperimental and clinical myocardial tissue engineering as a goldstandard.

Example 2 Electrophysiological Evaluation of the Elastomeric ScaffoldMembranes

Measurements of Electrical Impedance

Electrical Conduction

Myocardial electrical impedance has shown to be an effective indicatorof myocardial tissue characteristics and electrode tissue interface.Significant modifications have been demonstrated during tissue ischemia.

Electrical resistivity (also known as specific electrical resistance orvolume resistivity) is a measure of how strongly a material opposes theflow of electric current. A low resistivity indicates a material thatreadily allows the movement of electrical charge. The SI unit ofelectrical resistivity is the ohm [Ω].

Material and Methods

Pieces of microporous membranes of polyethyl acrylate (PEA100 in whatfollows) and a copolymer of ethyl acrylate and hydroxyethyl acrylatewith a 90:10 mass ratio of both monomers (hereafter PEA90) wereemployed. The membranes had been cut into pieces of dimensions 25 mm×25mm, with an approximate thickness of 1.0 mm (PEA100 and PEA90A) and of0.7 mm (PEA90B). The pores of the membranes consisted in layers oforthogonal families of parallel cylindrical pores, with pore diameter of150 microns and pore separation of 300 microns. They were generated byletting the precursors of the polymers react inside a template of theporous structure, and dissolving the template afterwards.

Scaffolds Pre-hydration

Elastomeric scaffolds need 2 days of pre-hydration as follows: 24 Hsimmersion in a PBS solution and 24 Hs immersion in culture medium.Vacuum could be necessary to improve tissue hydration, putting thesample in tube with cap and performing vacuum with a syringe. Once pHchange is observed, the samples should be overnight in fresh culturemedium.

Electrophysiological Studies

Two electrodes having curved needles for easy insertion were suturedinto the opposites borders of the elastomeric scaffolds and of 3Dcollagen type I matrix (n=5 for each sample). These electrodes wereconceived to be implanted for temporary postoperative cardiac pacing inheart surgery. Scaffolds and implanted electrodes were immersed in Petridishes containing DMEM cell culture medium. After 30 minuteselectrophysiological studies were performed connecting the electrodes toa Pacing System Analyzer Model 5311 (Medtronic Inc.). Bipolar chargebalanced electrostimulation was delivered using the followingparameters: pulse amplitude 1 Volt, pulse width 0.5 ms, frequency ofstimulation 70 pulses per minute (ppm). Electrostimulation was deliveredjust for testing. Afterwards electrical impedance within the scaffoldswas assessed.

Results

Electrical measurements were performed in each preparation group, i.e.cell medium alone, collagen matrix, PEA90A scaffold, PEA90B scaffolds,PEA100 scaffolds. Each group consisted of 5 samples.

Impedance measurements showed the following values: cell culture medium292+/−25 ohms, collagen matrix 230+/−21 ohms; PEA90A scaffolds 321+/−34Ohms; PEA90B scaffolds 345+/−33 ohms; PEA100 scaffolds 340+/−29 ohms

Cell Collagen PEA90A PEA90B PEA100 medium matrix scaffold scaffoldscaffold Impedance [Ω] 292 230 321 345 340 Current [mA] 3.42 4.35 3.112.90 2.94

Pacing pulse; 1.0 V, 0.5 ms

These results showed that all the evaluated materials present electricalconduction properties, i.e. resistance, similar to those encounteredwith cardiac tissues, thus these scaffolds have the potential to be usedfor myocardial substitution.

Example 3

The failing cardiac muscle needs to be chronically supported to decreaseventricular wall stress and also to be regenerated to improveventricular function. This Example demonstrates that the association ofstem cells with a collagen matrix and a polyester mesh for cardiac wrapprovides better results than the implantation of polyester mesh alone.

To illustrate this embodiment, fifteen sheep underwent 1 hour ofsurgical myocardial ischemia followed by reperfusion. Three groups werecreated: Group 1: coronary occlusion without treatment (control group).Group 2: LV constraint using a polyester mesh for cardiac wrap. Group 3:the ischemic area was treated associating stem cells, a collagen matrixand a polyester mesh. Autologous adipose tissue derived stem cells (ASC)cultured in hypoxic conditions (5%) were labelled with BrdU and injectedinto the infarct area and into a collagen matrix. At 3 months animalswere evaluated with echocardiography and histopathological studies.

Biopsy Extraction

In 15 female Rambouillet sheep weighing 32 to 37 kg (mean 35±2.2 Kg),subcutaneous fat tissue was removed for stem cell isolation andexpansion. Autologous cells were used throughout in order to avoid anyproblem of histocompatibility. Adipose tissue biopsies were obtained bysubcutaneous fat tissue removal (40-60 grams) from the right thoracicwall and stored in phosphate buffered saline (PBS) at room temperatureuntil processing.

Isolation and Hypoxic Culture of Adipose Derived Stem Cells (ASC)

The tissue samples were finely minced and digested by incubation in a0.14 Wünsch units/mL Liberase Blendzyme 2 (Roche Applied Science,Hvidovre, Denmark) solution at 37° C. for two hours. The digests werecentrifuged at 400 g for 10 min and the top fluid and fat layers werediscarded. Contaminating erythrocytes were lyzed by resuspension of thepellet in sterile milli-Q water for 20 seconds, after which the saltconcentration was adjusted through addition of 10×PBS. The cells werefiltered through a 100 μm cell strainer, centrifuged at 400 g for 10min, and resuspended in 25 mL growth medium, consisting of minimumessential medium alpha (A-MEM) (GIBCO/Invitrogen) supplemented with 10%fetal bovine serum (FBS), and penicillin (10 U/ml), streptomycin (10mg/ml), gentamicin (10 mg/ml) (all from GIBCO/Invitrogen). The cellswere seeded in a T75 flask and transferred to a CO₂ incubator overnight,after which non-adherent cells were removed. The flasks were thentransferred to a hypoxic workbench/incubator (Xvivo; Biospherix, Lacona,N.Y.), allowing for uninterrupted cell culture and passaging in acontrolled atmosphere of 5% O₂ and 5% CO₂ balanced with nitrogen. Duringexpansion of the cells, the media was changed twice a week. When cellswere 90% confluent, the cells were detached from the culture flasksusing 0.125% trypsin/0.01% EDTA and transferred to new flasks.

Labeling with Bromodeoxyuridine

For each sample, the cells were expanded until eight T175 culture flaskswere 75% confluent, then the cells were labeled with bromodeoxyuridine(BrdU). Briefly, cells were incubated with growth media containing 10micrograms BrdU (Sigma) for 48 hours, and then the cells were washedseveral times with PBS and frozen in aliquots of approximately 10×10⁶cells.

Experimental Myocardial Injury

After preoperative medication and induction of anaesthesia (sameprotocol as fat tissue biopsies) animals were intubated and mechanicallyventilated with an Aestiva/5 system (Datex-Ohmeda, Helsinki, Finland).The electrocardiogram was monitored during operation. A central venousline was placed through the external jugular vein for administration offluid and drug infusions. Left thoracotomy was performed at the level ofthe 5th intercostal space, and the heart was exposed. To reduce the riskof ventricular fibrillation, a continuous IV perfusion (2 mg/kg perhour) of Xylocaine 1% (Lidocaine, AstraZeneca) was performed during theentire surgical procedure. In all animals a LV myocardial ischemia wassurgically created by transitory ligation (60 minutes) of the maindiagonal branch of the left coronary artery, followed by reperfusion. A4-0 non-absorbable Prolene suture was passed underneath the coronaryartery branch, the flow was interrupted using a Teflon pledgetcompressed by a polyurethane occluder. This occluder was released after60 minutes, thus the myocardial ischemic territory was reperfused.Significant EKG changes, including widening of the QRS complex andelevation of the ST segment, and colour and kinetics changes of the areaat risk were considered indicative of coronary occlusion.

Treatment Groups

Animals were randomized in 3 groups:

Group 1 (n=5): myocardial ischemia without treatment (control group).

Group 2 (n=5): post-ischemia implantation of a mesh ventricular wrapdevice.

Group 3 (n=5): post-ischemia intrainfarct injection of stem cells,implantation of a collagen matrix as interface and implantation of themesh ventricular wrap device.

Cell Injection and Collagen Matrix Implantation

In Group 3 animals, at 1 hour of infarction cell were injected into theinfarct zone by using a 27-gauge needle. Injections consisted of 99+/−12million cells, 50% (2 mL) injected into the infarction and 50% (5 mL)seeded into a 3D collagen type I matrix.

For myocardial treatment, six injection needle points were used in eachanimal, bulging over the myocardial infarction area was confirmed inevery case after injection. Criteria to guide the epicardial injectionswere the ventricular surface discoloration and hypokinesia.

Collagen Matrix Preparation

Collagen matrix was prepared from a commercially available CE Markcollagen kit (Pangen 2, Urgo Laboratory, Chenove, France). This 3Dbiodegradable matrix (size: 5×7×0.6 cm) was manufactured using alyophilised, non denatured, native type I collagen. The matrix poresmeasured 50 to 100 μm. In the operating room and under high sterilityconditions, matrix was placed into a Petri dish; afterward, the cellsuspension (50±6 million cells diluted in 5 mL medium) was seeded ontothe matrix. To promote a regular distribution of ASC into the matrixpores, Petri dishes containing the collagen matrix was shakencontinuously for 10 minutes at 160 g using an Orbital Shaker (StuartScientific, Stone, Staffordshire, UK).

Mesh Cardiac Wrap

To avoid hemodynamic instability and arrhythmias during implantation, westart to fix the mesh cardiac wrap (C or Cap polyester device) beforecreation of myocardial ischemia. The C or Cap model Gen2 CSD Size B(Acorn Cardiovascular Inc, St Paul, Minn., USA) was chosen in all cases,then was opened longitudinally, slid behind the ventricles and fixedwith 2 lateral epicardial sutures (Prolene 4-0). Afterwards the ischemiawas created followed by reperfusion. One hour later the cells wereinjected, the collagen matrix implanted and the anterior part of the Cor Cap was closed using a continuous suture (Prolene 2-0). The fixationof the device was completed by multiple single sutures over theatrio-ventricular anterior groove.

Results

No mortality was observed. The hypoxic treatment for cell culturesdemonstrated a quite dramatic improvement of proliferation rate: underhypoxia cells grown faster. Echocardiography showed a limitation of LVED(Left Ventricular End-diastolic Dimension) volume in both treated groups(polyester mesh alone 35.6±5 mL and with cell therapy 32.6±4 mL) vs.control (65±6.3 mL, p<0.01 for both comparisons). EF (Ejection Fraction)was significantly greater in the hearts treated with the polyester mesh+cells/collagen (55.8±3.8%) compared with those receiving polyesterwrapping-only (44.1±2.3%) (p=0.04) or without treatment (34.8±3.6%)(p=0.01). Doppler-derived mitral valve deceleration time (DT) improvedfrom 140±6.3 ms to 195±9.5 ms (p=0.03) in the cell-collagen C or Capgroup but not in the other groups. Histology showed in the cell treatedgroup multifocal ischemic areas much less prominent than in othergroups, with focuses of angiogenesis and viable grafted cells. Minimalfibrosis interface between the polyester mesh and the epicardium wasobserved in Group 3, probably due to the interposition of thecell-seeded collagen.

Comments

In an ischemic model, stem cells associated with a collagen matrix and apolyester mesh for cardiac wrapping improves EF and diastolic function,reducing adverse remodelling and fibrosis. This procedure associating aregenerative biological approach with a prosthetic support device seemsto be appropriate for the treatment of advanced ischemic heart failure.

Example 4

This clinical Example demonstrates that a cell-seeded collagen matrixassociated with intra-infarct cell therapy provides better results thanstem cell alone.

Matrix Preparation

A 3D biodegradable matrix (size: 5×7×0.7 cm) manufactured usinglyophilized bovine type I collagen was prepared. The matrix poresmeasured 50 to 100 μm. In the operating room and under high sterilityconditions, matrix was placed into a Petri dish; afterward, the cellsuspension (250±28 million cells diluted in 10 ml medium) was seededonto and into the matrix. To promote a regular distribution of cellsinto the matrix, Petri dishes containing the matrix were shakencontinuously for 10 minutes at 160 g using an Orbital Shaker (StuartScientific, Stone, Staffordshire, UK).

Surgical Procedure

In 10 patients (mean age 52.6 y), after sternotomy, a single OP-CABG(off pump-coronary artery bypass graft) was performed using the leftinternal mammary artery (LIMA). At the end of surgery, 250±28 millioncells diluted in 5 ml medium were injected in the infarcted area andborderzone, using a 25G×40 mm retrobulbar ophthalmic needle. Then thecell seeded matrix was placed covering the infarcted area and fixed tothe epicardium with 6 PDS sutures (6-0).

In another group of 10 patients (mean age 56.8 y), a single OP-CABG wasperformed. Stem cells were injected into the infarction scar but noseeded matrix was used in this group.

Results

There was no mortality and any related adverse events (follow-up 10±3.5months). NYHA FC improved in both groups from 2.3±0.5 to 1.3±0.5(matrix, p=0.0002) vs 2.4±0.5 to 1.5±0.5 (no matrix, p=0.001). LVend-diastolic volume evolved from 142.4±24.5 to 112.9±27.3 mL (matrix,p=0.02) vs 138.9±36.1 to 148.7±41 mL (no matrix, p=0.57), LV fillingdeceleration time improved significantly in the matrix group from 162±7ms to 198±9 ms (p=0.01) vs no-matrix group (from 159±5 ms to 167±8 ms,p=0.07). Scar area thickness progress from 6±1.4 to 9±1.1 mm (matrix,p=0.005) vs 5±1.5 to 6±0.8 mm (no matrix, p=0.09). EF improved in bothgroups, from 25.3±7.3 to 32±5.4% (matrix, p=0.03) versus 27.2±6.9 to34.6±7.3% (no matrix, p=0.031).

Comments

This clinical study showed that cell transplantation associated with acollagen cell-seeded matrix increased the thickness of the infarct scarwith viable tissues and help to normalize cardiac wall stress in injuredregions (scaffold effect), thus limiting ventricular remodelling andimproving diastolic function. Patients treated without the cell-seededcollagen matrix didn't show limitation of post ischemic remodelling andimprovements in diastolic function.

Example 5 Preparation of an Hybrid Material for Three-DimensionalCulture with Improved Mechanical Properties Filling ElastomericMembranes with Self-Assembling Synthetic Peptides Resuspended in Water

Mechanical Properties of Three Dimensional Scaffolds

During the last decades cellular cardiomyoplasty has become a state ofart for cardiac affects. It consists in introducing myocardial or stemcells (with and without three-dimensional matrices) in the infarctedventricles trying to recover the lost function. The drawback is that itwas proved that there were a low number of cells capable of surviving inthese conditions; partly because they cannot stand the mechanical forcesof the receptor tissue.

Three-dimensional scaffolds as RAD16-I (self-assembling peptidesresuspended in water) allow the cells to form a functional network inthe β-sheet scaffold formed, but additionally it is indispensable thatthe scaffold could stand the beat of the heart. Elastomeric membranescan offer these mechanical properties.

Congo Red Staining

Congo red staining is a simple method to appreciate the formation oftypical RAD16-I self-assembly β-sheet. The reactive is as sodium salt ofbenzidinediazo-bis-1-naphthylamine-4-sulfonic acid (formula:C₃₂H₂₂N₆Na₂O₆S₂) and its configuration permits hydrogen bonding of theazo and amine groups of the dye to similarly spaced hydroxyl radicalsgiving a red coloration.

Material and Methods

Pieces of microporous membranes of polyethyl acrylate (PEA100 in whatfollows) and a copolymer of ethyl acrylate and hydroxyethyl acrylatewith a 90:10 mass ratio of both monomers (hereafter PEA90) were employedas elastomeric membranes. The membranes had been cut into pieces ofdimensions 0.5 cm×0.5 cm, with an approximate thickness of 1.0 mm(PEA100 and PEA90). The pores of the membranes consisted in layers oforthogonal families of parallel cylindrical pores, with pore diameter of150 microns and pore separation of 300 microns. They were generated byletting the precursors of the polymers react inside a template of theporous structure, and dissolving the template afterwards.

The self assembling peptide RAD16-I was used as three-dimensionalscaffold. The stock was prepared in 1% solution of sucrose 10% avoidingthe self-assembling produced by the increase of the ionic strength. Thestock solution is diluted to the desired concentration in sucrose 10%for each experiment.

Scaffolds Pre-hydration

The elastomeric scaffolds needed to be pre-conditioned before thepeptide addition. Initially the membranes were sterilized using threewashes with EtOH 70% and letting them dry in air during 10 min. Afterthis pre-treatment the scaffolds were hydrated as follows: 30 min ofimmersion in a PBS solution with vacuum and three washes with sucrose10%. The vacuum was necessary to ensure that all the pores were filledwith the aqueous solution, and the isotonic solution was necessary toavoid the self-assembly during the first contact between the membranesand the peptide. After this treatment, the membranes were dried to moistsince the complete drying would return the membranes to their initialhydrophobicity.

Filling of the Membranes with RAD16-I Peptide

The pre-treated membranes were introduced inside a 9-mm-diameter cellculture insert (PICM01250, Millipore, Billerica, Mass.). Then RAD16-Ipeptide 0.15% in sucrose 10% was loaded, carefully, on the top of themembrane using a micropipette. After the loading of the peptide, 500 μLof DMEM complete medium with L-glutamine, sodium pyruvate and 15% FetalBovine Serum was placed out of the insert. The peptide was let toself-assemble in the flow cabinet during 20 min. At this point themedium penetrates in the insert from the bottom membrane inducing abottom-to-top self-assembly of RAD16-I inside the membrane. To wash outthe remaining sucrose, medium was added in sequential steps on the topof the ensemble and allowed to infiltrate. Finally 500 μL were loadedinside the insert and 2.5 mL in the well outside the insert.

Results and Comments

Both, PEA100 and PEA90 membranes are filled with RAD16-I peptide. Eachgroup consisted on 2 samples. It is therefore considered a compositematerial: elastomeric membranes+self-assembling peptides.

The results showed that PEA100 membrane allows RAD16-I to fill theporous easily than PEA90 membrane. Thus, it seems that the mosthydrophobic PEA100 polymer is in principle preferable in order to obtainthe combined system with improved mechanical properties compared withthose of the peptide gel, that would permit to hold the heartbeat.

Example 6

The goal of this example is to show the viscoelasticity evaluation ofelastomer matrix scaffolds for ventricular support and myocardialregeneration.

Viscoelastic properties of myocardial tissue has been recentlyidentified as a major determinant of contraction and relaxationcoupling. The goal of our approach is to develop tissue engineeredimplants for ventricular support and myocardial regeneration, usingnanobiomaterials associated with stem cell grafting. In the presentstudy viscoelastic properties of several nanobiopolymers developed wereassessed by applying a constant stress. Their stress-strain responses aswell as their temporal dependencies mimicked the behavior of theclassical Kelvin Standard Linear Solid Model which combines a Voigtsystem (hookean spring E2 in parallel with a viscous dashpot n2), and ahookean spring (E1) in series of the Voigt system. Thus, under aconstant stress, the materials instantaneously deform to some strain,which is the elastic part of the strain, and after that it will continueto deform and asymptotically approach a steady-state strain. This lastpart is the viscous component of the strain.

Methods

We evaluated viscous and elastic properties of 3 types of porousmembranes:

A) a non-degradable copolymer of ethyl acrylate and hydroxyethylacrylate with a 90:10 mass ratio of monomers (PEA 90),

B) partially degradable polymer: methacrylate-endcapped caprolactone(CLMA) membranes, and

C) native collagen matrices of bovine origin (control group).

The elastic modulus E1 and E2 (in mN/mm) and the viscosity coefficient(n2 in mN/mm s) were calculated using the load clamp technique. Piecesof 12 mm×1 mm, thickness 1 mm of PEA 90 matrices were studied in water(8 days) (1), in water (1 hour) (2) or only in air (3); same protocolfor CLMA matrices: (4), (5), (6); and same protocol for collagenmatrices: (7), (8), (9).

Results

All samples showed linear stress-strain relationship, simplifying theevaluation of viscoelasticity. In all groups, E1 ranged from 20 to 40mN/mm, except in (6) where E1 was about 180 mN/mm. In all groups, E2ranged from 10 to 100 mN/mm, except in (6) where E1 was about 400 mN/mmIn all groups, n2 ranged from 1 to 5 mN/mm s, except in (6) where n2 wasabout 18 mN/mm.s. All studied matrices exhibited a viscoelastic behaviorsimilar to the Kelvin Standard Linear Solid Model. However, allviscoelastic coefficients E1, E2 and n2 were higher in CLMA in air thanin all other groups.

Conclusions

Viscous and elastic properties of “bioactive implants” of the presentinvention match the characteristics of myocardial contraction andrelaxation activity at both structural and functional bio integrativelevels. Bioactive implants specially conditioned for the recovery ofleft and/or right ventricular myocardium may reduce adverse chamberremodelling and fibrosis.

Example 7 Tensile Properties of Partially Degradable Polymer MatricesEmployed for the Fabrication of the Membranes

This example aims to give representative information about the swellingcapacity and the tensile mechanical properties of three possible polymercompositions employed for the elastomer membrane fabrication, as afunction of their weight fraction of degradable and non-degradableparts.

Polyethylacrylate (PEA) was chosen as the non-degradable polymer, andpoly(caprolactonemethacryloyloxyethyl ester) (PCLMA) as the partiallydegradable polymer. Three systems containing both polymers were preparedby radical polymerization of the monomers in mass ratios of 15:85, 50:50and 85:15 (see table). Sheets of the three bulk polymers of 0.3 mmthickness were obtained in this way, and samples of dimensions 0.3 mm×30mm×6 mm were cut from those sheets in order to perform mechanical tests.The table gives, for each system prepared, the weight fraction ofnon-degradable part, of partially degradable component, and ofdegradable part.

Stress-strain measurements were made in tensile mode in a MicrotestSCM3000 95 apparatus by stretching the specimen at a constant strainrate of 0.01/min and simultaneously measuring the force applied to thespecimen. The tests were continued until the samples broke. The testswere carried out on samples equilibrated in phosphate buffer saline(PBS). At least 3 replicates of each sample were tested. The weightincrease after 48 h immersion in PBS was referred to the dry weight ofthe sample to define the “swelling mass increase”.

TABLE Tensile and swelling properties of the three polymer matrices.mass ratio of partially mass ratio mass ratio degradable swelling strainSystem of of non- polymer mass breaking at Young PEA:PCLMA degradabledegradable (PCLMA) increase strength break modulus mass ratio part (%)part (%) (%) (%) (KPa) (%) (Pa) 15:85 40.1 59.9 85 6.83 ± 0.15 1982 ±117 19.8 ± 1.8 11961 ± 1295 50:50 23.6 76.4 50 5.32 ± 0.13 1904 ± 40223.7 ± 5.0 10214 ± 1368 85:15 7.1 92.9 15 1.92 ± 0.40 3445 ± 299 55.8 ±0.8 9816 ± 472

It is to be understood that the above-described embodiments areillustrative of only a few of the many possible specific embodiments,which can represent applications of the principles of the invention.Numerous and varied other arrangements can be readily devised inaccordance with these principles by those skilled in the art withoutdeparting from the spirit and scope of the invention.

What is claimed is:
 1. A bioactive implant constituting a scaffold formyocardial regeneration and ventricular chamber support, comprising: I.an elastomeric microporous membrane made from a mix of at least onenon-degradable polymer and at least one partially degradable polymer,said membrane having a porosity comprised between 70% and 90%, the poresbeing interconnected and having diameters comprised between 50 micronsand 500 microns, wherein (a) Said non-degradable polymer is selectedfrom the group consisting of polyethylacrylate and polyethylacrylatecopolymerized with a 10% wt or a 20% wt hydroxyethylacrylate comonomer;and (b) Said partially degradable polymer is selected from the groupconsisting of caprolactone 2-(methacryloyloxy)ethyl ester andcaprolactone 2-(methacryloyloxy)ethyl ester copolymerized withethylacrylate' in weight proportions of this last comonomer comprisedbetween 30% and 80%, wherein a percentage of non-degradable polymersversus degradable polymers is comprised between 10% wt and 90% wt, II. ananofiber hydrogel and III cells.
 2. The bioactive implant of claim 1,wherein said nanofiber hydrogel comprises: molecules selected from thegroup consisting of protein, peptide, oligosaccharide, polysaccharide,proteoglycan derived matrices such as collagens, fibrins, alginates,chitosans, and hyaluronic acid; and/or synthetic molecules of peptideAcN-RADARADARADARADA-COONH2.
 3. The bioactive implant of claim 1,wherein said cells are myogenic cells or cardiomyogenic cells.
 4. Thebioactive implant of claim 3, wherein the cells are selected from thegroup consisting of skeletal myoblasts, smooth muscle cells, fetal andneonatal cardiomyocytes, adult ventricular cardiomyocytes, cardiospheresand epicardial progenitors.
 5. The bioactive implant of claim 1, whereinsaid cells are angiogenic cells.
 6. The bioactive implant of claim 5,wherein the cells are selected from the group consisting of bone marrowand peripheral blood mononuclear fraction, bone marrow and peripheralblood endothelial progenitors, endothelial cells, mesothelial cells fromomentum, adipocyte derived stem cells, stem cells from adiposeepicardial tissue and multipotent menstrual blood stromal cells.
 7. Thebioactive implant of claim 1, wherein said cells are pluripotent stemcells.
 8. The bioactive implant of claim 7, wherein the cells areselected from the group consisting of animal embryonic cells, adult stemcells, fetal stem cells, neonatal stem cells, non-human stem cells,umbilical cord cells, induced pluripotent stem cells (iPSCs),bone-marrow mesenchymal stem cells (MSCs), adult testis pluripotent stemcells and human amniotic fluid stem cells (hAFSCs).
 9. The bioactiveimplant of claim 1, wherein the elastomeric microporous membrane issurface-treated to graft adhesion molecules selected from the groupconsisting of functional peptides of RGD peptides, functional sugars orlipids, and proteins of laminin or laminin fragments.
 10. The bioactiveimplant of claim 1, wherein the nanofiber hydrogel is a natural polymerselected from the group consisting of collagen, alginate, chitosan, selfassembling peptide, hyaluronic acid and fibrin.
 11. The bioactiveimplant of claim 1, wherein the composition of the bioactive implant hasa percentage of non-degradable versus degradable polymer, which rangesfrom 10% wt to 48% wt.
 12. The bioactive implant of claim 1, furthercomprising pacing electrodes for synchronous electrostimulation orelectrophysiological treatments.
 13. The bioactive implant of claim 12wherein the electrostimulation or electrophysiological treatments aredefibrillation and resynchronization.
 14. The bioactive implant of claim1, further comprising labels for evaluation of biodegradation,integration, proliferation, differentiation and/or function, usingradiologic, ultrasound-echocardiographic, radioisotopic, metabolic, RMI,CT Scan or bio-luminescence-fluorescence methods.
 15. The bioactiveimplant of claim 1, wherein the labels are selected from dies,microspheres, radioisotopes, and iron-particles.
 16. The bioactiveimplant of claim 1, further comprising a system for the controlledrelease or absorbance of active molecules selected from the groupconsisting of small organic molecules, peptides, lipids, sugars,proteins, and proteoglycans having angiogenic, antiangiogenic,pro-regenerative, anti-regenerative, apoptotic, necrotic, antiapoptoticand antinecrotic activity.
 17. The bioactive implant of claim 1 furthercomprising active molecules selected from the group consisting of VEGF,IL-6, IL-10, IGF-1,FGF-2, HBEGF and bFGF, and chitosan.
 18. Thebioactive implant of claim 1, further comprising cytokines andangiogenic antiapoptotic peptides.
 19. The bioactive implant of claim 1,having one or two helical loops of at least one non-degradable polymer,wherein the implant has a conical shape.